Faculty of Life Sciences, The University of Manchester, Oxford Road, Manchester, UK, M13 9PT.
Plant Methods. 2009 Oct 28;5:14. doi: 10.1186/1746-4811-5-14.
The progress and completion of various plant genome sequencing projects has paved the way for diverse functional genomic studies that involve cloning, modification and subsequent expression of target genes. This requires flexible and efficient procedures for generating binary vectors containing: gene fusions, variants from site-directed mutagenesis, addition of protein tags together with domain swaps and deletions. Furthermore, efficient cloning procedures, ideally high throughput, are essential for pyramiding of multiple gene constructs.
Here, we present a simple, flexible and efficient PCR-fusion/Gateway cloning procedure for construction of binary vectors for a range of gene fusions or variants with single or multiple nucleotide substitutions, short sequence insertions, domain deletions and swaps. Results from selected applications of the procedure which include ORF fusion, introduction of Cys>Ser mutations, insertion of StrepII tag sequence and domain swaps for Arabidopsis secondary cell wall AtCesA genes are demonstrated.
The PCR-fusion/Gateway cloning procedure described provides an elegant, simple and efficient solution for a wide range of diverse and complicated cloning tasks. Through streamlined cloning of sets of gene fusions and modification variants into binary vectors for systematic functional studies of gene families, our method allows for efficient utilization of the growing sequence and expression data.
随着各种植物基因组测序项目的进展和完成，为涉及目标基因克隆、修饰和随后表达的各种功能基因组研究铺平了道路。这需要灵活高效的程序来生成包含基因融合、定点突变变体、添加蛋白标签以及结构域交换和缺失的二元载体。此外，对于多个基因构建体的级联，高效的克隆程序（理想情况下是高通量）是必不可少的。
在这里，我们提出了一种简单、灵活和高效的 PCR 融合/Gateway 克隆程序，用于构建一系列带有单个或多个核苷酸取代、短序列插入、结构域缺失和交换的基因融合或变体的二元载体。该程序的一些应用结果得到了证明，包括 ORF 融合、引入 Cys>Ser 突变、插入 StrepII 标签序列以及拟南芥次生细胞壁 AtCesA 基因的结构域交换。
所描述的 PCR 融合/Gateway 克隆程序为广泛的各种复杂克隆任务提供了一种优雅、简单和高效的解决方案。通过将基因融合和修饰变体的集合克隆到二元载体中，用于对基因家族进行系统的功能研究，我们的方法允许有效地利用不断增长的序列和表达数据。
